Process for identifying fish signals

ABSTRACT

Among embodiments is disclosed a process for immunostaining a sample such that the stain is stable under FISH conditions.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation application of U.S. patent application Ser. No.11/685,736, filed Mar. 13, 2007, which application claims benefit under35 U.S.C. §119(e) from U.S. Provisional Applications Ser. Nos.60/821,550 filed on Aug. 4, 2006 and 60/781,888 filed on Mar. 13, 2006.

BACKGROUND OF THE INVENTION

All references cited in this specification, and their references, areincorporated by reference herein where appropriate for teachings ofadditional or alternative details, features, and/or technicalbackground.

1. Field of the Invention

The present invention includes a method for combining immunostaining andFISH using covalently binding small molecule tags.

2. Description of the Related Art

Many methods are known to aid in the microscopic analysis of samples.For example, without limitation, it is known that certain dyes have anaffinity for certain cellular structures. Such dyes may therefore beused to aid in analysis by helping to further elucidate such structures.

Fluorescence microscopy of cells and tissues is well known in the art.Methods have been developed to image fluorescent cells in a microscopeand extract information about the spatial distribution and temporalchanges occurring in these cells. Some of these methods and theirapplications are described in an article by Taylor, et al. in AmericanScientist 80 (1992), p. 322-335. These methods have been designed andoptimized for the preparation of a few specimens for high spatial andtemporal resolution imaging measurements of distribution, amount andbiochemical environment of the fluorescent reporter molecules in thecells.

Detection of fluorescent signals may be by way of an epifluorescentmicroscope which uses emitted fluorescent light to form an image(whereas a conventional reflecting microscope uses scatteredillumination light to form an image). The excitation light of anepifluorescence microscope is used to excite a fluorescent tag in thesample causing the fluorescent tag to emit fluorescent light. Theadvantage of an epifluorescence microscope is that the sample may beprepared such that the fluorescent molecules are preferentially attachedto the biological structures of interest thereby allowing identificationof such biological structures of interest.

The acronym “FISH” references a technique that uses chromophore tags(fluorophone) that emits a secondary signal if illuminated with an lightto detect a chromosomal structure. FISH uses fluorescent probes whichbind only to those parts of the chromosome with which they show a highdegree of sequence similarity. Such tags may be directed to specificchromosomes and specific chromosome regions. The probe has to be longenough to hybridize specifically to its target (and not to similarsequences in the genome), but not too large to impede the hybridizationprocess. Typically, the probe is tagged directly with fluorophores. Thiscan be done in various ways, for example nick translation or PCR usingtagged nucleotides. If signal amplification is necessary to exceed thedetection threshold of the microscope (which depends on many factorssuch as probe labelling efficiency, the kind of probe and thefluorescent dye), secondary antibodies or streptavidin are bound to thetag molecules, thus amplifying the signal.

The FISH technique may be used for identifying chromosomal abnormalitiesand gene mapping. For example, a FISH probe to chromosome 21 permits oneto “fish” for cells with trisomy 21, an extra chromosome 21, the causeof Down syndrome. FISH kits comprising multicolor DNA probes arecommercially available. For example, AneuVysion Multicolor DNA Probe Kitsold by the Vysis division of Abbott Laboratories, is designed for invitro diagnostic testing for abnormalities of chromosomes 13, 18, 21, Xand Y in amniotic fluid samples via fluorescence in situ hybridization(FISH) in metaphase and interphase nuclei. The AneuVysion Assay (CEP 18,X, Y-alpha satellite, LSI 13 and 21) Multi-color Probe Panel uses CEP18/X/Y probe to detect alpha satellite sequences in the centromereregions of chromosomes 18, X and Y and LSI 13/21 probe to detect the13q14 region and the 21q22.13 to 21q22.2 region. The combination ofcolors evidenced is used to determine whether there is normal chromosomenumbers or trisomy. In a similar vein, the UroVysion kit by the Vysisdivision of Abbott Laboratories is designed to detect chromosomalabnormalities associated with the development and progression of bladdercancer by detecting aneuploidy for chromosomes 3, 7, 17, and loss of the9p21 locus via fluorescence in situ hybridization (FISH) in urinespecimens from persons with hematuria suspected of having bladdercancer.

Another process for detecting structures of interest is immunostaining.Immunostaining refers to the laboratory process of detecting biologicalmaterials using antibodies. Often these antibodies are labeled with afluorescent compound which can be viewed by a microscope. Antibodiesthat detect, for example, a protein of interest in the biological sampleare generated by a foreign host species (a polyclonal antibody) orcultured immune cell clones (monoclonal antibodies). After exposure tothe foreign protein, the antibodies can be harvested and used as veryspecific and sensitive detection agents. Antibodies so generated areknown as “primary antibodies,” as they bind directly to the protein ofinterest. Certain immunostaining agents can be applied in a singlestage, where the primary antibody is directly linked to a colouringagent. In other cases, the primary antibody is targeted by a “secondary”antibody, targeting a species-specific part of the structure of theprimary antibody. The later technique may be advantageous in that thesignal is amplified, as multiple secondary antibodies will bind to aprimary antibody. It also allows for a high variety of primaryantibodies—researchers can make their own antibodies and not have toconjugate them to a colouring agent themselves. Finally, it means that avariety of colouring agents can be conjugated to any given species ofsecondary antibody, and are available in ready supply. This has openedthe door to “double-labelling” experiments, where several proteins canbe co-localised.

Traditionally the combination of immunostaining techniques with FISH hasbeen challenging. If immunostaining is performed first, subsequent FISHtreatment may abolish mostly non-covalent antibody-antigen interaction.Similarly, if FISH is performed first, the subsequent antibody treatmentmay release the FISH probe because of its low salt concentration. As theimmunostaining of certain portions of a biological sample as well as theFISH staining of other areas of the same biological sample may beadvantageous, it would be advantageous if a system allowing for combinedimmunostaining and FISH staining could be developed.

SUMMARY OF THE INVENTION

Embodiments disclosed herein include:

A method comprising in order: (a) treating a biological sample havingchromosomal material therein with one or more antibodies having anaffinity for at least one non-chromosomal portion of such biologicalsample, the antibody(ies) having an introduced non-detectable reactiveconjugate thereon; (b) treating said biological sample with afluorescently-tagged chromosome probe having a high degree of sequencesimilarity to one or more portions of said chromsomal material; and (c)treating said biological sample with a detectable tag reactive with saidnon-detectable reactive conjugate on said antibody(ies) but not withsaid chromosomal material or non-chromosomal portions of said biologicalsample. The non-detectable reactive conjugate may be a biontinylatedtyramide, and the detectable tag reactive therewith may be streptavidintagged with a fluorophore.

Alternatively, there is provide a method comprising in order: (a)treating a biological sample having chromosomic material therein withone or more antibodies having an affinity for at least onenon-chromosomal portion of such biological sample, the antibody(ies)having an introduced a detectable reactive conjugate thereon; (b)treating said biological sample with a fluorescently-tagged chromosomeprobe having a high degree of sequence similarity to one or moreportions of said chromosomic material; and (c) treating said biologicalsample with a non-detectable or detectable tag reactive with saiddetectable reactive conjugate on said antibody(ies) but not with saidchromosomic material or non-chromosomal portions of said biologicalsample.

Further provided is a method for fixing biological material to asurface, said method comprising the steps of: (a) obtaining a biologicalsample in an aqueous supernatant and placing a least a portion of saidsample on a surface to which part of the sample is to be fixed; (b)removing aliquot volumes of said supernatant replacing the same with asimilar volume of alkyl alcohol wherein said removal and replacementoccurs a plurality of times so as to gradually fix the part of sample tothe surface. The alkyl alcohol may be a C₁-C₁₂ alcohol, a C₁-C₆ alcohol,or methanol.

Yet further provided is a method for loading a density centrifugationgradient, said method comprising the steps of: (a) preparing acentrifugation gradient; (b) applying sample to said centrifugationgradient by means of a capillary funnel.

Also disclosed is a method for the simultaneous identification ofmultiple sub-cellular components, said method comprising the steps of:immunostaining a sample of cells with antibodies specific to each ofsaid sub-cellular components to be identified; simultaneously processingsaid sample of cells with one or more fluorescent in situ hybridizationprobes comprising distinct fluorophores to discriminate between each ofsaid sub-cellular components to be identified; visualizing andquantitating fluorescent signals produced by said probes in a microscopysystem.

Further disclosed is a process for identifying and enumeratingfluorescent in situ hybridization (“FISH”) signals produced with respectto nuclear components hybridized in situ with fluorescent markers:acquiring using an epi-fluorescence microscope a plurality of images atdifferent focal planes in each fluorescence channel corresponding to thehybridized FISH markers; selecting a best focused image from saidplurality of images for each nucleus; acquiring using saidepi-fluorescence microscope a plurality of images above and below thefocal plane of said best focused image for each nucleus; selecting foreach nucleus the one focal plane above and the one focal plane below thefocal plane of said best focused image in which the image is bestfocused; combining said images from the one focal plane above and belowsaid best focused image with said best focused image each nucleus toproduce a combined image for said nucleus; analyzing the combined imageof each nucleus to separate background pixels from signal pixels, and todetermine areas of produced signals corresponding to pre-set size andshape criteria corresponding to a non-art factual target.

DETAILED DESCRIPTION OF THE INVENTION

There is disclosed herein a number of techniques for marking subcellularcomponents of a cell to allow for identification of the same, andbiomedical decisions to be made based on images of the same.

In one embodiment subcellular component are stained using an immunostaincomprising antibodies specific to each of the sub-cellular components tobe identified, and tagging with one or more FISH probes comprisingdistinct fluorophores that discriminate between each of the sub-cellularcomponents to be identified. The signals produced are quantitated by anautomated microscopy system.

In one embodiment, there is disclosed a process of identifying andenumerating fluorescent in situ hybridization (“FISH”) signals producedwith respect to nuclear components hybridized in situ with fluorescentmarkers, the process comprising the steps of: (a) acquiring using anepi-fluorescence microscope a plurality of images at different focalplanes in each fluorescence channel corresponding to the hybridized FISHmarkers; (b) selecting a best focused image from said plurality ofimages for each nucleus; (c) acquiring using said epi-fluorescencemicroscope a plurality of images above and below the focal plane of saidbest focused image for each nucleus; (d) selecting for each nucleus theone focal plane above and the one focal plane below the focal plane ofsaid best focused image in which the image is best focused; (e)combining said images from the one focal plane above and below said bestfocused image with said best focused image to produce a combined imagefor said nucleus; (0 analyzing the combined image for each nucleus toseparate background pixels from signal pixels, and to determine areas ofproduced signals corresponding to a pre-set size and shape criteriacorresponding to a non-artifactual target.

In another embodiment there is provided a method for the simultaneousidentification of multiple sub-cellular components, the methodcomprising the steps of: (a) immunostaining a sample of cells withantibodies specific to each of the sub-cellular components to beidentified; (b) simultaneously process the sample of cells with one ormore fluorescent in situ hybridization probes comprising distinctfluorophores to discriminate between each of the subcellular componentsto be identified; and (c) visualizing and quantitating fluorescentsignals produced by the probes in a microscopy system.

The sub-cellular component may be any cell component. For example, thesub-cellular component may be one indicative of developmental age. Forexample, telomeric length may be determined from signals and used todetermine the age of the cell.

Testing may be performed on a host of microscope slides, and such slidesmay be optionally coded with digitally readable information whichdescribes the sample thereon or the test to performed on the sample.

For example, a microscope slide having a poly-L-lysine coating thereonalong at least an area wherein the sample is to be deposited on theslide may be utilized. A poly-L-lysine coating aids in cell, cellularmaterial, and other biological material adhesion to the slide.Application of the biological material to the slide portion coated withpoly-L-lysine may be aided by use of a walled chamber with at least apartially opened top and bottom portion, into which the material to bedeposited on the slide may be placed. The walled chamber may beconnected to a base which is operatively configured for holding amicroscope slide, which may be coded, and intercalated therewith (e.g.,in a tongue and groove configuration). The portion of the microscopeslide onto which the sample is placed can be positioned under the wallstructure in a manner such that a defined area of coverage is set. Thewalled chamber may be pinioned at a fixed point to allow the walledchamber to be pivoted up when the microscope slide is placed into orremoved from the base by pushing or pulling on the slide.

A biological material may also be fixed to a surface of the slide by amethod comprising the steps of (a) obtaining a biological sample in anaqueous supernatant and placing a least a portion of said sample on asurface to which part of the sample is to be fixed; (b) removing aliquotvolumes of said supernatant replacing the same with a similar volume ofalkyl alcohol wherein said removal and replacement occurs a plurality oftimes so as to gradually fix the part of sample to the surface. Thealkyl alcohol may be selected from the group consisting of: the alkylalcohol may be a C₁-C₁₂ alcohol, a C₁-C₆ alcohol, or methanol.

Identification and enumeration of fluorescent in situ hybridization(“FISH”) signals produced with respect to nuclear components hybridizedin situ with fluorescent markers may be by performed by a number ofdifferent methods. One method which may find use comprises the steps of:acquiring with an epi-fluorescence microscope a plurality of images atdifferent focal planes in each fluorescence channel corresponding to thehybridized FISH markers; selecting a best focused image from saidplurality of images for each nucleus; acquiring with saidepi-fluorescence microscope a plurality of images above and below thefocal plane of said best focused image for each nucleus; selecting foreach nucleus the one focal plane above and the one focal plane below thefocal plane of said best focused image in which the image is bestfocused; combining said images from the one focal plane above and belowsaid best focused image with said best focused image each nucleus toproduce a combined image for said nucleus; and analyzing the combinedimage for each nucleus to separate background pixels form signal pixels,and to determine areas of produced signals corresponding to a pre-setsize and shape criteria corresponding to a non-artifactual target.

In an embodiment method for detecting circulating nucleated fetal cells,blood may, for example, be transferred to a conical tube with volumeexpanded. The volume may then be mixed and added to prepared gradients.The density centrifugation gradient may be loaded manually by use of apipettor or alternatively, it has been found, by means of a disposableplastic capillary funnel that allows unassisted loading of gradients bygravity. Tubes may then be centrifuged with centrifuge brake turned offto prevent disruption of the gradient when slowing down. The nucleatedcells may be removed and may further be diluted, and then centrifugedonce again. After removal of the supernatant, the cells may beresuspended, for example in PBS.

Cells may then be deposited onto a microscope slide, such as apoly-L-lysine coated slide or slide chamber, as discussed above.Methanol followed by 2% formaldehyde in PBS (=phosphate buffered saline,e.g. at ph 7.4), after pouring off of the methanol, may be used to fixthe cells to the slide. The supernatant may be removed in aliquots, withintroduction of methanol gradually. Such technique avoids abrupt changesin solvent properties and may gradually fix target materials. After anymethanol/formaldehyde/PBS solution has been removed, the fixed cells maybe stored in PBST (=PBS with 0.05% Tween 20) until ready forimmunostaining.

In one immunostaining technique, the slides may be incubated withantibody, e.g., anti-mouse IgG-HRP (=peroxidase-conjugated RabbitAnti-Mouse IgG) conjugate and/or AntiHb_(E)-CRTX (Anti-hemoglobin(epsilon chain) monoclonal antibody). Follow-up staining with a DNAcomplexing agent, such as DAPI (4′-6-diamidino-2-phenylindole). whichforms fluorescent complexes with natural double-stranded DNA, may alsobe performed. Slides may be equilibrated in PBS, with a pepsin stocksolution being used to improve nuclei concentration. Nuclei may be fixedonto slides using, for example, a formaldehyde, MgCl₂ in PBS solutionfollowed by dehydration in an ethanol series and air drying.

Probe hybridization may then be undertaken. The probe may be thermallycycled on the slide, and the slides may then be placed into a rack asdescribed above and then placed in bulk into a humidified FISH chamberand allowed to hybridize. Non-specifically bound probe can then beremoved by methods known in the art. The probe treated sample may thenbe counterstained with a DNA stain, such as DAPI, dehydrated in anethanol series and air dried. The samples on the slides may then beimaged to determine binding of the antibody and DNA stain, and frombinding characteristics to determine the characteristics of the sample.

In one possible selection, the antibody may include a biotinylatedtyramide functionality which in itself is not detectable. After FISHtreatment, however, the antibody may be elucidated by using streptavidinlabeled with a fluorophore.

Statement Regarding Preferred Embodiments

While the invention has been described with respect to preferredembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the inventionwithout departing from the spirit or scope of the invention as definedby the appended claims.

1. A method comprising in order: (a) treating a biological sample havingchromosomal material therein with one or more antibodies having anaffinity for at least one non-chromosomal portion of such biologicalsample, the antibody(ies) having an introduced non-detectable reactiveconjugate thereon; (b) treating said biological sample with afluorescently-tagged chromosome probe having a high degree of sequencesimilarity to one or more portions of said chromosomic material; and (c)treating said biological sample with a detectable tag reactive with saidnon-detectable reactive conjugate on said antibody(ies) but not withsaid chromosomal material or non-chromosomal portions of said biologicalsample.
 2. A method comprising in order: (a) treating a biologicalsample having chromosomal material therein with one or more antibodieshaving an affinity for at least one non-chromosomal portion of suchbiological sample, the antibody(ies) having an introduced a detectablereactive conjugate thereon; (b) treating said biological sample with afluorescently-tagged chromosome probe having a high degree of sequencesimilarity to one or more portions of said chromosomic material; and (c)treating said biological sample with a non-detectable or detectable tagreactive with said detectable reactive conjugate on said antibody(ies)but not with said chromosomal material or non-chromosomal portions ofsaid biological sample.
 3. A method for fixing biological material to asurface, said method comprising the steps of: (a) obtaining a biologicalsample in an aqueous supernatant and placing a least a portion of saidsample on a surface to which part of the sample is to be fixed; (b)removing aliquot volumes of said supernatant replacing the same with asimilar volume of alkyl alcohol wherein said removal and replacementoccurs a plurality of times so as to gradually fix the part of sample tothe surface.
 4. The method of claim 3 wherein said alkyl alcohol isselected from the group consisting of: the alkyl alcohol may be a C₁-C₁₂alcohol, a C₁-C₆ alcohol, or methanol.
 5. A method for loading a densitycentrifugation gradient, said method comprising the steps of: (a)preparing a centrifugation gradient; (b) applying sample to saidcentrifugation gradient by means of a capillary funnel.
 6. A method forthe simultaneous identification of multiple sub-cellular components,said method comprising the steps of: immunostaining a sample of cellswith antibodies specific to each of said sub-cellular components to beidentified; simultaneously processing said sample of cells with one ormore fluorescent in situ hybridization probes comprising distinctfluorophores to discriminate between each of said sub-cellularcomponents to be identified; visualizing and quantitating fluorescentsignals produced by said probes using a microscopy system.
 7. A processfor identifying and enumerating fluorescent in situ hybridization(“FISH”) signals produced with respect to nuclear components hybridizedin situ with fluorescent markers: acquiring using an epi-fluorescencemicroscope a plurality of images at different focal planes in eachfluorescence channel corresponding to the hybridized FISH markers;selecting a best focused image from said plurality of images for eachnucleus; acquiring using said epi-fluorescence microscope a plurality ofimages above and below the focal plane of said best focused image foreach nucleus; selecting for each nucleus the one focal plane above andthe one focal plane below the focal plane of said best focused image inwhich the image is best focused; combining said images from the onefocal plane above and below said best focused image with said bestfocused image each nucleus to produce a combined image for said nucleus;analyzing the combined image of each nucleus to separate backgroundpixels from signal pixels, and to determine areas of produced signalscorresponding to pre-set size and shape criteria corresponding to anon-art factual target.